| Oodescelis chinensis Kaszab (Coleoptera: Tenebrionidae) is a Chinese endemic species,which is only distributed in desert and semidesert regions of Xinjiang, China. Oodescelischinensis possesses significant ecological and economic values, and is suitable to be used as amodel creature because of its obviously sexual dimorphism and the habit of not killing eachother. O. chinensis lives through winter on the surface of soil, and the insect can surviveafter supercooling point test experiment. It is a suitable creature for studying antifreezeproteins. But its biology and antifreeze proteins remain largely unknown. Also, its laboratoryrearing and its sexual dimorphism are yet to be determined. In this study, the laboratoryrearing methods, the biological characteristics and the sexing methods of O. chinensis wereexamined. Furthermore, eleven antifreeze protein genes were isolated from overwinter adults,and the structures and functions of three antifreeze proteins were studied. This study laidfoundation for further research of the desert insect.The Glass Petri dishes added with humid cotton were used to cultivate the insect eggs,prepupae, pupae and teneral adults. The larvae were reared in insect rearing tubes with moistdesert sand. The water content of desert sand decreased from bottom to upper part because ofcapillarity, and the surface of the desert soil was dry. The wheat bran was added to raise larvaeof O. chinensis. It can finish its life cycle under the rearing condition. The survival rates ofeggs, first instar larvae, larvae after second instar, prepupae, pupae, and teneral adults were86.39±4.43％,92.85±1.22,55.22±5.65％,82.35±4.37,85.13±3.41％and86.00±7.37％,respectively. Oodescelis chinensis can successfully finish its life cycle, and the insect can passto the next generation normally in laboratory.O. chinensis adopts nocturnality to survive desert environment. It had one-year life cycleand overlapping generation, in Urumqi. The female/male ratio of adult insect was1:1. Theweight and elytrum length of female was obviously greater than that of male in adult. Therewere15instars during larvae stage. The coloration of each developmental stage gradually changed from creamy white to light brownish, brunneus or black. The egg laying period offemale adult lasts4months. On average, each female produced1032eggs. The adults andpupae have abviously sexual dimorphism apparences.The adult of Oodescelis chinensis has three externally visible gender-specificcharacteristics, and an inner characteristic on the8thsternite for sex determination. First, thereare broad claw pads in tarsus of the male adult fore legs and middle legs. These claw padswere consisted of slim flat bands which can be seen with scanning electron microscope. Theforeparts of the slim flat bands are slightly sharp and the tips are bent back. The back of thestructure has bamboo-shaped screw threads. Second, the hind legs of male adult are coveredwith bristle layers. Third, the first, second and fifth abdominal segments of male adult eachhas one bristle pad. The fourth, there is a trilateral indentation on the8thabdominal sternite inmale. However, these characteristics in male are absent in female adult. The fouthcharacteristic is very common in tenebrionidae beetles, such as Microdera punctipennis,Colposcelis microderoides microderoides, Anatolica polita borealis, Adesmia anomaladejeani, Sternoplax souvorowiana and Anatolica pseudiduma. A new method based on thedifference of the8thsternite was developed to determinate the sexes of desert beelte adults.This method could be widely used in sexing desert beeltes with100%accuracy and withoutdemage. The pupaes of O. chinensis also has three sexual characteristics. First, similar to maleadults, the tarsus of the male pupal fore legs and middle legs are obviously wider than that offemale, which will be developed to claw pads. Second, there are no obviously genitalopenings on the8thabdominal sternite in male pupae. Third, the8thabdominal sternite of malepupae is narrower than that of female. There are two closed prominencies at the end of the8thabdominal sternite in male pupae, but the two prominencies of female pupae are separated andlooked like nipples. The second and the third characteristic of pupae are universal inTenebrionidae beetle pupae, so it can be used in pupal sex determination of tenebrionidi beetlewithout damage. The claw pads in tarsus and indentation on the8thabdominal sternite of Oodescelis chinensis male adults are helpful during coupling and mating.Eleven antifreeze genes were isolated from antifreeze tolerant desert beetle O. chinensisin this research. The primary structure of these protein comprised of tandem repeats of12-aasequence [TCT(I)xSxxCxxAx] with regularly spaced Cys at intervals of6-aa residues, andthere are23-aa signal peptides in the N-terminal part of these antifreeze proteins. The genesof these antifreeze proteins can be divided into two types. One is the antifreeze genes thatcode primary structure of TCT(I)xSxxCxxAx repeated sequence. These type genes includecoding122-aa,94(95)-aa and83-aa antifreeze protein genes. The other is the gene that codeprimary structure of TCIxSxxCxxAx repeated sequence, which only codes97-aa. Theantifreeze genes Ocafp1-3(122-aa), Ocafp2(97-aa) and Ocafp4(95-aa) were selected toconstructe with different expressive plasmids (pET28a-egfp and pET32a), and thecorresponding mature peptide has been expressed in E. coli, respectively. The recombinantantifreeze proteins EGFP-OcAFPs and TrxA-OcAFPs were obtained after purification byNi-IDA. The structions and functions of these recombination antifreeze proteins are asfollows. The secondary structures of these antifreeze proteins are mainly β-sheet which isconfirmed by circular dichroism spectrometer. The results of differential scanning calorimetryshowed that OcAFP1-3and OcAFP4has moderate thermal hysteresis activity (THA) and pHand heat stability, but they can‘t endure tempersture higher than60℃. The THAof OcAFP2istoo low to be measured. TGA themogravimetric analysis suggests that the hydrophilicity ofOcAFP4is stronger than that of OcAFP1-3, and the hydrophilicity of OcAFP2is the weakestamong these antifreeze proteins. The results observed with nanolitre osmometer showed thatthe modification ice crystal of OcAFP1-3, OcAFP4and OcAFP2are hexagon, quadrilateraland bone nails shape. OcAFP1-3and OcAFP4made ice crystal growing along a-axis, andOcAFP2made ice crystal growing along c-axis. These phenomena indicate that OcAFP1-3and OcAFP4are mainly combined to the basal plate of the ice crystal, and OcAFP2is mainlycombinated to the prism faces of the ice crystal. Our results indicate that OcAFP1-3and OcAFP4have both characteristics of antifreeze protein with high and moderate activities;while OcAFP2has characteristics of antifreeze protein with moderate and low activities.These new discoveries shed light on our understanding of the antifreeze proteins obtainedfrom freeze tolerant insect. |
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